High-concentration monoclonal antibody formulations

ABSTRACT

The present application discloses high-concentration monoclonal antibody formulations suitable for subcutaneous administration, e.g. via a pre-filled syringe. In particular, it discloses a formulation comprising a spray dried monoclonal antibody at a concentration of about 200 mg/mL or more suspended in a non-aqueous suspension vehicle where the viscosity of the suspension vehicle is less than about 20 centipoise. Also disclosed are: a subcutaneous administration device with the formulation therein, a method of making the formulation, a method of making an article of manufacture comprising the suspension formulation, use of the formulation in the preparation of a medicament, and a method of treating a patient with the formulation.

This non-provisional application filed under 37 CFR § 1.53(b), claimsthe benefit under 35 USC § 119(e) of U.S. Provisional Application Ser.No. 61/649,146, filed on May 18, 2012, which is incorporated byreference in entirety.

FIELD OF THE INVENTION

The present invention concerns high-concentration monoclonal antibodyformulations suitable for subcutaneous administration, e.g. via apre-filled syringe. In particular, the invention concerns a formulationcomprising a spray dried monoclonal antibody at a concentration of about200 mg/ml, or more suspended in a non-aqueous suspension vehicle,wherein the viscosity of the suspension vehicle is less than about 20centipoise. The invention also concerns a subcutaneous administrationdevice with the formulation therein, a method of making the suspensionformulation, a method of making an article of manufacture comprising thesuspension formulation, use of the suspension formulation in thepreparation of a medicament, and a method of treating a patient with thesuspension formulation.

BACKGROUND OF THE INVENTION

Outpatient administration of high-dose monoclonal antibodies (several mgper kg) via subcutaneous (SC) injection is a preferred form of deliveryfor treating chronic conditions (Stockwin and Holmes, Expert Opin BiolTher 3:1133-1152 (2003); Shire et al., J Pharm Sci 93:1390-1402 (2004)).The subcutaneous route of administration that requires injections usingsyringes, auto-injectors, or other devices generally restricts productformulation with regards to injection volume and solution viscosity, anddevice functionalities in terms of injection force and time. To deliverhigh-dose of monoclonal antibody with limitations of injection time,volume, and force, a high-concentration monoclonal antibody formulation(100 mg/mL or greater) is required for subcutaneous administration(Stockwin and Holmes, Expert Opin Biol Ther 3:1133-1152 (2003); Shire etal., J Pharm Sci 93:1390-1402 (2004)). A potential challenge in thedevelopment of high protein concentration formulations isconcentration-dependent solution viscosity. Injection force (or glideforce) is a complex factor influenced by solution viscosity, the size ofthe needle (i.e., needle gauge), and surface tension ofcontainer/closure. Smaller needles, e.g., ≥26 gauge, will pose less painsensation to the patients. Overcashier and co-workers established aviscosity-glide force relationship as a function of needle gauge basedon Hagen-Poiseuille Equation (Overcashier et at, Am. Pharm Rev.9(6):77-83 (2006)). With a 27-gauge thin walled (TW) needle (ID, min.:0.241 mm), the liquid viscosity should be maintained below 20 centipoisein order not to exceed the glide force of 20 newton. Unfortunately,formulation scientists are constantly challenged against a conflictingreality with high monoclonal antibody concentration and high solutionviscosity (Shire et al., J Pharm Sci 93:1390-1402 (2004); Kanai et al.,J Pharm Sci 97:4219 4227 (2005)). Another challenge with liquidformulations at high monoclonal antibody concentration is proteinphysical stability. Greater aggregation rates and undesirableopalescence are generally observed in high monoclonal antibodyconcentration liquid solutions (Alford et al., J Pharm Sci 97:3005-3021(2008); Salinas et al., J Pharm Sci 99:82-93 (2010); Sukumar et al.,Pharm Res 21:1087-1093 (2004)).

Different formulation strategies have been attempted to reduce theviscosity of high-concentration monoclonal antibody liquid solution byformulating with salt, amino acid, or sugar to balance repulsive andattractive forces through intermediate ionic strengths (Sukumar et al.,Pharm Res 21:1087-1093 (2004); He et al., J Pharm Sci 100:1330-1340(2011)). However, the effectiveness of these approaches may be limitedat monoclonal antibody concentration beyond 100 mg/mL or due to specificcharacteristics of certain monoclonal antibodies. Dani and co-workersapplied the approach of reconstituting spray-dried monoclonal antibodypowder to prepare high monoclonal antibody concentration liquid solutionprior to subcutaneous injection (Dani et al., J Pharm Sci 96:1504-1517(2007)). This approach can certainly improve the protein stability inthe solid state during the entire shelf life, however the high viscosityissue still remains because the spray dried monoclonal antibody powderneeds to be reconstituted at high monoclonal antibody concentrationprior to injection. A powder-based approach emerged recently usingmonoclonal antibody crystalline particle suspensions (Yang et al., ProcNatl Acad Sci 100:6934-6939 (2003); Trilisky et al., “Crystallizationand liquid-liquid phase separation of monoclonal antibodies andFc-fusion proteins: Screening results,” AICHE online publication DOI 10,1002/btrp.621 (published by Wiley Online Library) (2011)). It is basedon the perception that viscosity of a crystal monoclonal antibodysuspension may be lower than a liquid formulation at the same monoclonalantibody concentration. However, no viscosity or injection force datawere presented in these references and this concept remainedspeculative. Furthermore, monoclonal antibody crystallization is not yeta mature process platform applicable to a wide range of monoclonalantibodies although some successful examples have been presented(Trilisky et al., “Crystallization and liquid-liquid phase separation ofmonoclonal antibodies and Fc-fusion proteins: Screening results,” AICHEonline publication DOI 10, 1002/btrp.621 (published by Wiley OnlineLibrary) (2011)).

The present invention represents a different powder-based conceptemploying a high-concentration monoclonal antibody powder suspension ina non-aqueous suspension vehicle. The suspension approach has beencomprehensively reviewed (Floyd and Jain, “Injectable emulsions andsuspensions,” In: Pharmaceutical Dosage Forms: Disperse Systems Volume 2(eds. Lieberman H A, Rieger M M, Banker G S). Dekker, NY, N.Y., p261-318(1996); Akers et al., J Parent Sci & Techn 41:88-96 (1987)) and has beenreported for microsphere/emulsion suspensions in vegetable oils, such assesame oil (Larsen et al., Eur J Pharm Sci 29:348-354 (2006); Hirano etal., J Pharm Sci 71:495-500 (1982)), soybean oil (Salmerón et al., DrugDev Ind Pharm 23:133-136 (1997); Karasulu et al., Drug Dev 14:225-233(2007)), and peanut oil (Santucci et al., J Contr Rel 42:157-164 (1996))as parenteral injectables. The physical and chemical forces influencingthe properties of non-aqueous suspensions can be quite different fromthose of aqueous suspension due to the absence of electrical effectsassociated with the DLVO theory (van der Waals attraction andelectrostatic repulsion as the result of double layer of counterions).

Pena and co-workers (Pena et al., Intl J Pharm 113:89-96 (1995))reported rheological characterization of excipient-free bovinesomatotropin (rbSt) powder (lyophilized or spray-dried) suspension incaprylic/capric triglyceride (MIGLYOL 812®) oil with or withoutpolysorbate 80. RbSt is a 191-amino acid peptide with a molecular weightof 22,000 daltons. Pena et al. determined that a network formed amongdrug particle, polysorbate 80, and MIGLYOL 812®, and a higher viscositywas observed with increasing polysorbate 80 and powder concentrations.These studies also found that particle shape/morphology played animportant role in suspension viscosity. The smaller spherical (moredensely packed) spray-dried particles resulted in more viscoussuspensions than the lyophilized counterpart which displayed largerirregular shaped flakes.

The non-aqueous powder-based approach for high concentration monoclonalantibody concentration suspensions remains unexplored. Studies with thesmall rbSt peptide in Pena et al. would not predict the ability toeffectively formulate a large tetrameric monoclonal antibody (about150,000) daltons). In addition, the oil vehicles used by Pena et al.were too viscous to be considered for use in pre-filled syringeadministration. The viscosity of MIGLYOL 812®, sesame oil, soybean oil,peanut oil are ˜30 centipoise (cP) at 25° C., 43 cP at 25° C., 50 cP at25° C. and 35 cP at 37° C., respectively. In addition, Pena et al.determined the suspension performance of spray dried powder was inferiorto lyophilized counterpart.

Publications describing monoclonal antibody formulations include: U.S.Pat. No. 6,284,282 (Maa et al.); U.S. Pat. Nos. 6,267,958 and 6,685,940(Andya et al.); U.S. Pat. No. 6,171,586 (Lam et al.); U.S. Pat. Nos.6,875,432 and 7,666,413 (Liu et al.); WO2006/044908 (Andya et al.);US-2011-0076273-A1 (Adler et al.); US 2011/0044977 and WO 2011/012637(Adler et al.); US 2009/0226530A1 (Lassner et al.); US-A 2003/0190316(Kakuta et al.); US-A 2005/0214278 and US-A 2005/0118163 (Mizushima etal.); US-A 2009/0291076 (Morichika et al.); and US-A 2010/0285011(Imaeda et al.)

SUMMARY OF THE INVENTION

The objectives of the present study were to: (1) identify processparameters that dictate suspension performance; (2) assess thefeasibility of establishing monoclonal antibody powder suspensions(i.e. >250 mg monoclonal antibody/mL) with acceptable injectability(i.e. injection force ≤20 N through 27-gauge thin-walled (TW) needle)and physical suspension stability; and/or (3) understand the mechanismof suspension performance. To prepare monoclonal antibody powders, spraydrying was used. Spray drying is a mature, scalable, and efficientmanufacturing process. The short-term effect of spray drying onmonoclonal antibody was studied at accelerated temperature. An importantcriterion for suspension vehicle selection was that the viscosity of thesuspension vehicle be below 10 centipoise (cP). The three modelsuspension vehicles, propylene glycol dicaprylate/dicaprate, benzylbenzoate, and ethyl lactate, tested in this study have low viscosity andmet this requirement.

Inverse gas chromatography (IGC) has been used for surface energyanalysis (SEA) (Newell et al., Pharm Res 18:662-666 (2001); Grimsey etal., J Pharm Sci 91:571-583 (2002); Newell and Buckton, Pharm Res21:1440-1444 (2004); Saleem and Smyth, Drug Devel & Ind Pharm34:1002-1010 (2008); Panzer and Schreiber, Macromolecules 25:3633-3637(1992)). In IGC, a probe is injected into a column packed with thepowder of interest (stationary phase) and the time required for theprobe to pass through the column (t_(r)) is a measure of the magnitudeof the interaction between the probe and the stationary phase. Surfaceenergy can normally be divided into polar and dispersive (non-polar)components. Thus, the use of non-polar (alkanes) and polar (electronacceptor-donor or acid-base solvents) probes allowed these two surfaceenergy components to be quantified. Surface energies of the spray-driedparticles may serve as a more direct and relevant indicator tosuspension performance than other particle characteristics. Anotherparameter is heat of sorption which is a direct measure of the strengthof the interactions between a solid and gas molecules adsorbed on thesurface (Thielmann F., “Inverse gas chromatography: Characterization ofalumina and related surfaces,” In “Encyclopedia of Surface and ColloidScience Volume 4 (edit by P. Somasundaran). CRC Press, Boca Raton, Fla.,p3009-3031 (2006); Thielmann and Butler, “Heat of sorption onmicrocrystalline cellulose by pulse inverse gas chromatography atinfinite dilution,” Surface Measurement Services Application Note 203(http://www.thesorptionsolution.comllnforination_Application_Notus_IGC.php#Aps)(2007)). The IGC method was employed to measure the heat of sorptionbetween spray dried particles and the suspension vehicle in this study.

The experimental data herein demonstrate that the objectives wereachieved, and high-concentration monoclonal antibody suspensionformulations suitable for subcutaneous administration were developed.

Thus, in a first aspect, the invention concerns a suspension formulationcomprising a spray dried monoclonal antibody at a concentration of about200 mg/mL or more suspended in a non-aqueous suspension vehicle, whereinthe viscosity of the suspension vehicle is less than about 20centipoise.

In another aspect, the invention concerns a suspension formulationcomprising a spray dried full length human IgG1 monoclonal antibody at aconcentration from about 200 mg/mL to about 400 mg/mL suspended in anon-aqueous suspension vehicle with a viscosity less than about 20centipoise, wherein the formulation has an average particle size fromabout 2 microns to about 10 microns, and injection glide force less thanabout 15 newton.

The invention further concerns a subcutaneous administration device(e.g. a pre-filled syringe) with the formulation therein.

In another aspect, the invention concerns a method of making asuspension formulation comprising suspending a spray dried monoclonalantibody in a non-aqueous suspension vehicle with a viscosity less thanabout 20 centipoise, wherein the antibody concentration in thesuspension formulation is about 200 mg/mL or more.

Additionally, the invention provides a method of making an article ofmanufacture comprising filling a subcutaneous administration device withthe formulation herein.

In related aspects, the invention concerns use of the formulation in thepreparation of a medicament for treating a patient in need of treatmentwith the monoclonal antibody in the formulation, as well as a method oftreating a patient comprising administering the formulation to a patientin need of treatment with the monoclonal antibody in the formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Antibody stability (as size exclusion chromatography (SEC) %monomer change from right after spry drying) as a function of storagetime at 40° C. for bevacizumab/trehalose formulation spray-dried (

) and freeze dried (∘) as well as for trastuzumab/trehalose formulationspray dried (

) and freeze dried (∩).

FIG. 2: The viscosity-powder concentration profiles for propylene glycoldicaprylate/dicaprate suspensions with three monoclonal antibody (mAb)powders spray dried with a pilot-scale or a bench-top spray dryer:bevacizumab by pilot-scale (⋄), bevacizumab by bench-top (

), trastuzumab by pilot-scale (□), trastuzumab by bench-top (

), rituximab by pilot-scale (Δ), rituximab by bench-top (

), empirical fitting (solid line), and theoretical fitting from Equation4 (dash line).

FIG. 3: The glide force-mAb concentration profiles for rituximab powdersuspension in propylene glycol dicaprylate/dicaprate (Δ), ethyl lactate(⋄), benzyl benzoate (∘) and predicted glide force for mAb liquidsolution extracted from FIG. 4 in Overcashier et al. Am. Pharm Rev.9(6): 77-83 (2006) (

).

FIG. 4: The profiles of viscosity-mAb concentration for rituximab powdersuspension in propylene glycol dicaprylate/dicaprate (Δ), in benzylbenzoate (⋄), and in ethyl lactate (∘).

FIG. 5: Particle size distribution of rituximab suspensions in propyleneglycol dicaprylate/dicaprate (⋄), in benzyl benzoate (□), and in ethyllactate (Δ).

FIGS. 6A-C: Photographs of rituximab suspension at 150 mg/mL in ethyllactate after 2-week storage (6A), in ethyl lactate vortexed after 1-daystorage (6B), and in propylene glycol dicaprylate/dicaprate after 2weeks storage (6C). (Note: the tape is not part of the suspension butused for optical focusing during photo taking.)

FIGS. 7A and 7B: Rituximab suspensions. FIG. 7A: Particle sizedistribution of rituximab

suspensions in mixtures of propylene glycol dicaprylate/dicaprate andethyl lactate at 100/0 (⋄), 75/25 (

), 50/50 (□), 25/75 (

), and 0/100 (Δ). FIG. 7B: Photograph of rituximab suspension in 75/25propylene glycol dicaprylate/dicaprate/ethyl lactate mixture after2-week storage. (Note: the tape is not part of the suspension but usedfor optical focusing during photo taking.)

FIGS. 8A-1, 8A-2 and 8B provide the amino acid sequences of the heavychain (SEQ ID No. 1) and light chain (SEQ ID No. 2) of rituximabantibody. Each of the framework regions (FR) and each of thecomplementarity determining region (CDR) regions in each variable regionare identified, as are the human gamma 1 heavy chain constant sequenceand human kappa light chain constant sequence. The variable heavy (VH)region is in SEQ ID No. 3. The variable light (VL) region is in SEQ IDNo. 4. The sequence identifiers for the CDRs are: CDR H1 (SEQ ID No. 5),CDR H2 (SEQ ID No. 6), CDR H3 (SEQ ID No. 7), CDR L1 (SEQ ID No. 8), CDRL2 (SEQ ID No. 9), and CDR L3 (SEQ ID No. 10).

FIGS. 9A and 9B provide the amino acid sequences of the heavy chain (SEQID No. 11) and light chain (SEQ ID No. 12) of bevacizumab antibody. Theend of each variable region is indicated with ∥. The variable heavy (VH)region is in SEQ ID No. 13. The variable light (VL) region is in SEQ IDNo. 14. Each of the three CDRs in each variable region is underlined.The sequence identifiers for the CDRs are: CDR H1 (SEQ ID No. 15), CDRH2 (SEQ ID No. 16), CDR H3 (SEQ ID No. 17), CDR L1 (SEQ ID No. 18), CDRL2 (SEQ ID No. 19), and CDR L3 (SEQ ID No. 20).

FIGS. 10A and 10B provide the amino acid sequences of the heavy chain(SEQ ID No. 21) and light chain (SEQ ID No. 22) of trastuzumab antibody.The end of each variable region is indicated with ∥. The variable heavy(VH) region is in SEQ ID No. 23. The variable light (VL) region is inSEQ ID No. 24. Each of the three CDRs in each variable region is boxed.The sequence identifiers for the CDRs are: CDR H1 (SEQ ID No. 25), CDRH2 (SEQ ID No. 26). CDR H3 (SEQ ID No. 27), CDR L1 (SEQ ID No. 28). CDRL2 (SEQ ID No. 29), and CDR L3 (SEQ ID No. 30).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the active agent(e.g. monoclonal antibody) to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. Such formulations are sterile. Inone embodiment, the pharmaceutical formulation is suitable forsubcutaneous administration.

“Pharmaceutically acceptable” with respect to an excipient in apharmaceutical formulation means that the excipient is suitable foradministration to a human patient.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

“Subcutaneous administration” refers to administration (of aformulation) under the skin of a subject or patient.

A “stable” formulation is one in which the active agent (e.g. monoclonalantibody) therein essentially retains its physical stability and/orchemical stability and/or biological activity upon suspension and/orstorage. Preferably, the formulation essentially retains its physicaland chemical stability, as well as its biological activity uponsuspension and storage. The storage period is generally selected basedon the intended shelf-life of the formulation. Various analyticaltechniques for measuring protein stability are available in the art andare reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent LeeEd., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991); and Jones, A.Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. In oneembodiment, stability of the suspension formulation is assessed aroundthe time the spray dried particles are suspended in the vehicle toproduce the suspension formulation. In one embodiment, stability can beevaluated when the formulation is held at a selected temperature for aselected time period. In one embodiment, monoclonal antibody stabilityis assessed by size distribution (percentage monomer, aggregation,and/or fragmentation) before and after spray drying (e.g. before andafter spray drying over 3-month storage under the acceleratedtemperature of 40° C.). In one embodiment, size distribution is assessedusing size exclusion chromatography-high performance liquidchromatography (SEC-HPLC). In one embodiment, the percentage monomerloss (as measured by SEC-HPLC) over 3 months is less than about 10%, forexample less than 5%, e.g. at accelerated temperature of 40° C. In oneembodiment, stability is assessed by evaluating suspension physicalstability, e.g. visual inspection of settling and/or particlesedimentation rate.

“Spray drying” refers to the process of atomizing and drying a liquid orslurry comprising a protein or monoclonal antibody using gas (usuallyair or nitrogen) at a temperature above ambient temperature so as toproduce dry powder particles comprising the protein or monoclonalantibody. During the process, liquid evaporates and dry particles form.In one embodiment, the spray drying is performed using a spray dryer,e.g. which has an air inlet temperature from about 100° C. to about 220°C. and an air outlet temperature from about 50° C. to about 100° C.Particles can be separated from the gas by various methods such ascyclone, high pressure gas, electrostatic charge, etc. This definitionof spray drying herein expressly excludes freeze drying or crystallizingthe monoclonal antibody.

A “dry” particle, protein, on monoclonal antibody herein has beensubjected to a drying process such that its water content has beensignificantly reduced. In one embodiment, the particle, protein, ormonoclonal antibody has a water content of less than about 10%, forexample less than about 5%, e.g., where water content is measured by achemical titration method (e.g. Karl Fischer method) or a weight-lossmethod (high-temperature heating).

For the purposes herein, a “pre-spray dried preparation” refers to apreparation of the monoclonal antibody (usually a recombinantly producedmonoclonal antibody which has been subjected to one or more purificationsteps) and one or more excipients, such as stabilizers (e.g.saccharides, surfactants, and/or amino acids) and, optionally, a buffer.In one embodiment the preparation is in liquid form. In one embodimentthe preparation is frozen.

A “suspension formulation” is a liquid formulation comprising solidparticles (e.g. spray dried monoclonal antibody particles) dispersedthroughout a liquid phase in which they are not soluble. In oneembodiment, the solid particles in the suspension formulation have anaverage particle diameter from about 2 to about 30 microns, e.g. fromabout 5 to about 10 microns (e.g. as analyzed by laser diffraction).Optionally, the solid particles in the suspension formulation have apeak (highest percentage) particle size of less than about 30 micron,and optionally less than about 10 microns (e.g. as analyzed by laserdiffraction). The suspension formulation may be prepared by combiningspray dried monoclonal antibody particles with a non-aqueous suspensionvehicle. In one embodiment, the suspension formulation is adapted for,or suitable for, subcutaneous administration to a subject or patient.

As used herein “non-aqueous suspension vehicle” refers to apharmaceutically acceptable liquid which is not water-based and in whichspray dried monoclonal antibody particles can be suspended in order togenerate a suspension formulation. In one embodiment, the vehiclecomprises a liquid lipid or fatty acid ester or alcohol (e.g. propyleneglycol dicaprylate/dicaprate), or other organic compound such benzylbenzoate or ethyl lactate. The vehicle herein includes mixtures of twoor more liquids, such as a mixture of propylene glycoldicaprylate/dicaprate and ethyl lactate. Preferably, the non-aqueoussuspension vehicle has a viscosity (at 25° C.) of less than about 20centipoise (cP), optionally less than about 10 cP, and, in oneembodiment, less than about 5 cP. Examples of non-aqueous suspensionvehicles herein include the vehicles in the Table 1 below:

TABLE 1 Exemplary Non-Aqueous Suspension Vehicles and Their ViscosityVehicle Viscosity (cP) Ethanol 1.3 (25° C.) Dimethyl sulfoxide 2.0 (20°C.) N-methyl-2-pyrrolidone 1.66 (25° C.) Acetone 0.33 (20° C.) Benzylbenzoate 9 (25° C.) Tetrahydrofurfuryl alcohol 6.2 (25° C.) dimethylether of diethylene 1.2 (15° C.) glycol (Diglym) Ethyl lactate 2 (20°C.) Ethyl oleate 7.4 (20 ° C.) Isopropyl Myristate 5.7 (20° C.)Propylene glycol dicaprylate/dicaprate 9 (25° C.) (MIGLYOL 840 ®)

“Viscosity” refers to the measure of the resistance of a fluid which isbeing deformed by either shear stress or tensile stress; it can beevaluated using a viscometer or rheometer. Unless indicated otherwise,the viscosity measurement (centipoise, cP) is that at about 25° C.Viscosity as used herein can refer to that of either the non-aqueoussuspension vehicle per se or that of the suspension formulation.

“Injectability” refers to the ease with which the suspension formulationcan be administered to a subject. According to one embodiment of theinvention, the injectability of a given suspension formulation can besuperior to the injectability of a liquid formulation comprising thesame monoclonal antibody concentration and the same excipient(s) andconcentration(s) thereof. In one embodiment, injectability refers to theinjection glide force.

“Injection glide force” as used herein refers to the force required forthe injection of a solution at a given injection rate via a needle ofpredetermined gauge and length. In one embodiment, it is evaluated usingpre-filled syringe (e.g. 1.0 mL-long syringe with ≤25 gauge needle, orpreferably ≤27 gauge needle) with glide force analyzed and establishedas a function of the distance of the plunger rod travelling inside thesyringe at a steady compression rate (e.g. using “Syringe Glide ForceMeasurement” as in the Example herein). Time and force required for amanual injection (or time required for an injection using anautoinjector) may impact the usability of the product by the end-user(and thus compliance with the intended use of the product). In oneembodiment, the Hagen-Poiseuille equation is utilized to estimate thetravel (or glide) force (Equation 1).

$\begin{matrix}{F = {\frac{8\; Q\mspace{20mu} \mu \; L}{\pi \; R^{\hat{s}}} \times A}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

Q=Volumetric flow rateμ=Fluid viscosityL=Needle lengthR=Needle inner diameterA=Cross sectional area of syringe plungerF=Frictionless travel force

According to Equation 1, the glide force is dependent on a number ofparameters. The only parameter a formulation scientist can influence isviscosity. All other parameters (needle inner diameter, needle length,and cross sectional area of syringe plunger) are determined by thepre-fillable syringe itself. Formulations with a high viscosity can leadto high injection forces and long injection times since both parametersare proportional to viscosity. Generally accepted limits for injectionforce and injection time may depend e.g. on the indication and thedexterity of the patient population. In an embodiment exemplifiedherein, the parameters in Equation 1 were:

Q=Volumetric flow rate=0.1 mL/secondμ=Fluid viscosity=20 centipoiseL=Needle length=1.25 cmR=Needle inner diameter=0.0105 cm (27 gauge needle)A=Cross sectional area of syringe plunger=0.00316 cm²F=Frictionless travel force=16.6×10⁵ dyne=16.6 newton

In one embodiment, injection glide force is determined as a function ofmonoclonal antibody concentration by injecting 1-mL of suspensionformulation using a 1-mL long syringe through a 27-gauge thin walled(TW) staked needle in 10 seconds.

In one embodiment the injection glide force of the suspensionformulation is about 20 newtons or less.

In one embodiment the injection glide force of the suspensionformulation is about 15 newton or less.

In one embodiment the injection glide force is from about 2 newton toabout 20 newton.

In one embodiment the injection glide force is from about 2 newton toabout 15 newton.

In one embodiment the injection glide force is less than about 20newton.

In one embodiment the injection glide force is less than about 15newton.

As used herein. “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention (if used) generally has a pH from about 4.0 toabout 8.0, for example from about 5.0 to about 7.0, e.g. from about 5.8to about 6.2, and in one embodiment its pH is about 6.0. Examples ofbuffers that will control the pH in this range include acetate,succinate, succinate, gluconate, histidine, citrate, glycylglycine andother organic acid buffers. In one embodiment herein, the buffer is ahistidine buffer. A buffer is generally included in the pre-spray driedpreparation and may be present in the suspension formulation preparedtherefrom (but is not required therein).

A “histidine buffer” is a buffer comprising histidine ions. Examples ofhistidine buffers include histidine chloride, histidine acetate,histidine phosphate, histidine sulfate. In one embodiment, the histidinebuffer is histidine-acetate or histidine-HCl. In one embodiment, thehistidine buffer is at pH 5.5 to 6.5, optionally pH 5.8 to 6.2, e.g. pH6.0.

The term “excipient” refers to an agent that may be added to apreparation or formulation, for example: as a stabilizer, to achieve adesired consistency (e.g., altering the bulk properties), and/or toadjust osmolality. Examples of excipients herein include, but are notlimited to, stabilizers, sugars, polyols, amino acids, surfactants,chelating agents, and polymers.

A “stabilizer” herein is an excipient, or mixture of two or moreexcipients, which stabilizes a pharmaceutical formulation. For example,the stabilizer can prevent instability due to spray drying at elevatedtemperature. Exemplary stabilizers herein include saccharides,surfactants, and amino acids.

A “saccharide” herein comprises the general composition (CH2O)n andderivatives thereof, including monosaccharides, disaccharides,trisaccharides, polysaccharides, sugar alcohols, reducing sugars,nonreducing sugars, etc. Examples of saccharides herein include glucose,sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin,dextran, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol,mellibiose, melezitose, raffinose, mannotriose, stachyose, maltose,lactulose, maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc.The preferred saccharide herein is a nonreducing disaccharide, such astrehalose or sucrose.

Herein, a “surfactant” refers to a surface-active agent, preferably anonionic surfactant. Examples of surfactants herein include polysorbate(for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g.poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. In oneembodiment, the surfactant is polysorbate 20 or polysorbate 80. Thesurfactant may be included to prevent or reduce aggregation ordenaturation of the monoclonal antibody in the preparation and/orformulation.

The term “amino acid” as used herein denotes a pharmaceuticallyacceptable organic molecule possessing an amino moiety located atα-position to a carboxylic group. Examples of amino acids include:arginine, glycine, ornithine, lysine, histidine, glutamic acid,asparagic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine,tryptophane, methionine, serine, and proline. The amino acid employed isoptionally in the L-form. Examples of amino acids which can be includedas stabilizers in the preparations and/or formulations herein include:histidine, arginine, glycine, and/or alanine.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example. Specific examples of monoclonalantibodies herein include chimeric antibodies, humanized antibodies, andhuman antibodies.

A “spray dried” monoclonal antibody has been subjected to spray drying.The term includes the spray dried monoclonal antibody in powder form(i.e. prior to suspension) and in liquid form (i.e. when suspended inthe non-aqueous suspension vehicle to form the suspension formulation).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, so long as they exhibit the desired biologicalactivity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad.Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest hereininclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey, such as baboon, rhesus or cynomolgus monkey) and humanconstant region sequences (U.S. Pat. No. 5,693,780). An example of achimeric antibody herein is rituximab.

“Humanized” forma of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence, except for FR substitution(s) as noted above. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). Exemplary humanizedantibodies herein include trastuzumab and bevacizumab.

A “human antibody” herein is one comprising an amino acid sequencestructure that corresponds with the amino acid sequence structure of anantibody obtainable from a human B-cell. Such antibodies can beidentified or made by a variety of techniques, including, but notlimited to: production by transgenic animals (e.g., mice) that arecapable, upon immunization, of producing human antibodies in the absenceof endogenous immunoglobulin production (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807)); selection fromphage display libraries expressing human antibodies (see, for example,McCafferty et al., Nature 348:552-553 (1990); Johnson et al., CurrentOpinion in Structural Biology 3:564-571 (1993); Clackson et al., Nature,352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991);Griffith et al., EMBO J. 12:725-734 (1993); U.S. Pat. Nos. 5,565,332 and5,573,905); generation via in vitro activated B cells (see U.S. Pat.Nos. 5,567,610 and 5,229,275); and isolation from human antibodyproducing hybridomas. An example of a human antibody herein isofatumumab.

A “multispecific antibody” herein is an antibody having bindingspecificities for two or more different epitopes.

A “bispecific antibody” is an antibody with binding specificities fortwo different epitopes. An example of a bispecific antibody specificallycontemplated herein is HER3/EGFR Dual Acting Fab (DAF) molecule, such asDL11f comprising human IgG1 heavy chains (US 2010/0255010;WO2010/108127).

Antibodies herein include “amino acid sequence variants” with alteredantigen-binding or biological activity. Examples of such amino acidalterations include antibodies with enhanced affinity for antigen (e.g.“affinity matured” antibodies), and antibodies with altered Fc regione.g. with altered (increased or diminished) antibody dependent cellularcytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) (see,for example, WO 00/42072, Presta, L. and WO 99/51642, Iduosogie et al.);and/or increased or diminished serum half-life (see, for example,WO00/42072, Presta, L.).

An “affinity matured variant” has one or more substituted hypervariableregion residues of a parent antibody (e.g. of a parent chimeric,humanized, or human antibody) which improve binding of the affinitymatured variant.

The antibody herein may be conjugated with a “heterologous molecule” forexample to increase half-life or stability or otherwise improve theantibody. For example, the antibody may be linked to one of a variety ofnon-proteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol.

The antibody herein may be a “glycosylation variant” such that anycarbohydrate attached to its Fc region is altered. For example,antibodies with a mature carbohydrate structure that lacks fucoseattached to an Fc region of the antibody are described in US Pat Appl NoUS 2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa HakkoKogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine(GlcNAc) in the carbohydrate attached to an Fc region of the antibodyare referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No.6,602,684, Umana et al. Antibodies with at least one galactose residuein the oligosaccharide attached to an Fc region of the antibody arereported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju,S.) and WO 1999/22764 (Raju, S.) concerning antibodies with alteredcarbohydrate attached to the Fc region thereof. See also US 2005/0123546(Umana et al.) describing antibodies with modified glycosylation.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined. The CDRs of rituximab,bevacizumab, and trastuzumab are disclosed in FIGS. 8A-1, 8A-2, 8B,9A-B, and 10A-B, respectively.

A “full length antibody” is one which comprises an antigen-bindingvariable region as well as a light chain constant domain (CL) and heavychain constant domains, CH1, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof. Preferably, the fulllength antibody has one or more effector functions. In one embodiment, ahuman IgG heavy chain Fc region extends from Cys226, or from Pro230, tothe carboxyl-terminus of the heavy chain. However, the C-terminal lysine(Lys447) of the Fc region may or may not be present. Unless otherwisespecified herein, numbering of amino acid residues in the Fc region orconstant region is according to the EU numbering system, also called theEU index, as described in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. Rituximab, trastuzumab, andbevacizumab are examples of full length antibodies.

A “naked antibody” is a monoclonal antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety, polymer, orradiolabel. Rituximab, trastuzumab, and bevacizumab are examples ofnaked antibodies.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding, complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to differentclasses. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy chain constant domains that correspond to the differentclasses of antibodies are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.The antibody herein is a human IgG1 according to one embodiment of theinvention.

A “human IgG” antibody herein refers to full length antibody comprisinghuman IgG1 heavy chain constant domains.

The term “recombinant antibody” as used herein, refers to a monoclonalantibody (e.g. a chimeric, humanized, or human monoclonal antibody) thatis expressed by a recombinant host cell comprising nucleic acid encodingthe monoclonal antibody. Examples of “host cells” for producingrecombinant antibodies include: (1) mammalian cells, for example,Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0 and NS0cells), baby hamster kidney (BHK), Hela and Vero cells; (2) insectcells, for example, sf9, sf21 and Tn5; (3) plant cells, for exampleplants belonging to the genus Nicotiana (e.g. Nicotiana tabacum); (4)yeast cells, for example, those belonging to the genus Saccharomyces(e.g. Saccharomyces cerevisiae) or the genus Aspergillus (e.g.Aspergillus niger); (5) bacterial cells, for example Escherichia colicells or Bacillus subtilis cells, etc.

As used herein, “specifically binding” or “binds specifically to” refersto an antibody selectively or preferentially binding to an antigen.Preferably the binding affinity for antigen is of Kd value of 10⁻⁹ mol/lor lower (e.g. 10¹⁰ mol/l), preferably with a Kd value of 10¹⁰ mol/l orlower (e.g. 10⁻¹² mol/l). The binding affinity is determined with astandard binding assay, such as surface plasmon resonance technique(BIACORE®).

A “therapeutic monoclonal antibody” is a monoclonal antibody used fortherapy of a human subject. Therapeutic monoclonal antibodies disclosedherein include: CD20 antibodies for therapy of B cell malignancies (suchas non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or autoimmunediseases (such as rheumatoid arthritis and vasculitis); HER2 antibodiesfor cancer (such as breast cancer or gastric cancer); VEGF antibodiesfor treating cancer, age-related macular degeneration, macular edema,etc.

For the purposes herein, “rituximab” refers to an antibody comprisingthe variable heavy amino acid sequence in SEQ ID No. 3 and variablelight amino acid in SEQ ID No. 4, and, optionally, the heavy chain aminoacid sequence in SEQ ID No. 1 and light chain amino acid sequence in SEQID No. 2. This term specifically includes biosimilar rituximab.

For the purposes herein, “bevacizumab” refers to an antibody comprisingthe variable heavy amino acid sequence in SEQ ID No. 13 and variablelight amino acid in SEQ ID No. 14, and, optionally, the heavy chainamino acid sequence in SEQ ID No. 11 and light chain amino acid sequencein SEQ ID No. 12. This term specifically includes biosimilarbevacizumab.

For the purposes herein, “trastuzumab” refers to an antibody comprisingthe variable heavy amino acid sequence in SEQ ID No. 23 and variablelight amino acid in SEQ ID No. 24, and, optionally, the heavy chainamino acid sequence in SEQ ID No. 21 and light chain amino acid sequencein SEQ ID No. 22. This term specifically includes biosimilartrastuzumab.

The monoclonal antibody which is formulated herein is preferablyessentially pure and desirably essentially homogeneous (i.e. free fromcontaminating proteins etc). “Essentially pure” antibody means acomposition comprising at least about 90% by weight of the antibody,based on total weight of the composition, preferably at least about 95%by weight. “Essentially homogeneous” antibody means a compositioncomprising at least about 99% by weight of antibody, based on totalweight of the composition.

II. Monoclonal Antibodies to be Formulated Herein

Exemplary techniques for producing monoclonal antibodies which can beformulated according to the present invention follow. In one embodiment,the antigen to which the antibody binds is a biologically importantprotein and administration of the antibody to a mammal suffering from adisease or disorder can result in a therapeutic benefit in that mammal.However, antibodies directed against nonpolypeptide antigens (such astumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) arealso contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissue-type plasminogen activator(t-PA): bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta: enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted), human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF-b; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-b1, TGF-b2, TGF-b3, TGF-b4, orTGF-b5; a tumor necrosis factor (TNF) such as TNF-alpha or TNF-beta;insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-1(brain IGF-1), insulin-like growth factor binding proteins; CD proteinssuch as CD3, CD4. CD8, CD19, CD20, CD22 and CD40; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-alpha, -beta, and -gamma; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, TL-9 andIL-10; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2,HER3 or HER4 receptor; and fragments of any of the above-listedpolypeptides.

Exemplary molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22,CD34 and CD40; members of the ErbB receptor family such as the EGFreceptor, HER2, HER3 or HER4 receptor; B cell surface antigens, such asCD20 or BR3; a member of the tumor necrosis receptor superfamily,including DR5; prostate stem cell antigen (PSCA); cell adhesionmolecules such as LFA-1, Mac1, p150.95. VLA-4, ICAM-1, VCAM,alpha4/beta7 integrin, and alphav/beta3 integrin including either alphaor beta subunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF as well as receptors therefor;tissue factor (TF); a tumor necrosis factor (TNF) such as TNF-alpha orTNF-beta, alpha interferon (alpha-IFN); an interleukin, such as IL-8;IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor;mp1 receptor; CTLA-4; protein C etc.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

Exemplary antibodies which can be formulated according to the presentinvention include, but are not limited to the following: anti-ErbBantibodies, including anti-HER2 antibodies (e.g. trastuzumab orpertuzumab); antibodies that bind to a B-cell surface marker, such asCD20 (for example rituximab and humanized 2H7/ocrelizumab), CD22, CD40or BR3; antibodies that bind to IgE, including omalizumab (XOLAIR®)commercially available from Genentech, E26, HAEI, IgE antibody with anamino acid substitution at position 265 of an Fc region thereof (US2004/0191244 A1), Hu-901, an IgE antibody as in WO2004/070011, orantibody that binds the small extracellular segment on IgE, M1′ (e.g.47H4v5: see U.S. Pat. No. 8,071,097), see, also, Presta et al., J.Immunol. 151:2623-2632 (1993); International Publication No. WO95/19181; U.S. Pat. No. 5,714,338, issued Feb. 3, 1998; U.S. Pat. No.5,091,313, issued Feb. 25, 1992; WO 93/04173 published Mar. 4, 1993; WO99/01556 published Jan. 14, 1999; and U.S. Pat. No. 5,714,338;antibodies that bind to vascular endothelial growth factor (VEGF) (e.g.bevacizumab) or a VEGF receptor; anti-IL-8 antibodies (St John et al.,Chest, 103:932 (1993), and International Publication No. WO 95/23865);anti-PSCA antibodies (WO01/40309); anti-CD40 antibodies, including S2C6and humanized variants thereof (WO00/75348); anti-CD11a antibodies,including efalizumab (RAPTIVA®) (U.S. Pat. No. 5,622,700, WO 98/23761.Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant et al.,Transplantation 58:377-380 (1994)); anti-CD18 antibodies (U.S. Pat. No.5,622,700, issued Apr. 22, 1997, or as in WO 97/26912, published Jul.31, 1997); anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19,1998); anti-TNF-alpha antibodies including cA2 (REMICADE®) andadalimumab (HUMIRA®), CDP571 and MAK-195 (Afelimomab) (See, U.S. Pat.No. 5,672,347 issued Sep. 30, 1997, Lorenz et al. J. Immunol.156(4):1646-1653 (1996), and Dhainaut et al. Crit. Care Med.23(9):1461-1469 (1995)); anti-Tissue Factor (TF) (European Patent No. 0420 937 B1 granted Nov. 9, 1994); anti-human α4β₇ integrin (WO 98/06248published Feb. 19, 1998); anti-EGFR antibodies, including chimerized orhumanized 225 antibody as in WO 96/40210 published Dec. 19, 1996;anti-CD3 antibodies, such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7,1985); anti-CD25 or anti-tac antibodies such as CH1-621 (SIMULECT®) and(ZENAPAX®) (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); anti-CD4antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum39(1):52-56 (1996)); anti-CD52 antibodies such as alemtuzumab(CAMPATH-1H®) (Riechmann et al. Nature 332:323-337 (1988); anti-Fcreceptor antibodies such as the M22 antibody directed against FcγRI asin Graziano et al. J. Immunol. 155(10):4996-5002 (1995);anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkeyet al. Cancer Res. 55(23Suppl): 5935s-5945s (1995); antibodies directedagainst breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6(Ceriani et al. Cancer Res. 55(23): 5852s-5856s (1995); and Richman etal. Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bindto colon carcinoma cells such as C242 (Litton et al. Eur J. Immunol.26(1):1-9 (1996)); anti-CD38 antibodies. e.g. AT 13/5 (Ellis et al. J.Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195(Jurcic et al. Cancer Res 55(23 Suppl): 5908s-5910s (1995) and CMA-676or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al.Cancer Res 55(23 Suppl): 5899s-5907s (1995); anti-EpCAM antibodies suchas 17-1A (PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab orc7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®);anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such asPRO542; anti-hepatitis antibodies such as the anti-Hep B antibodyOSTAVIR®; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitopeantibody BEC2; anti-αvβ3 antibody VITAXIN®; anti-human renal cellcarcinoma antibody such as ch-G250; ING-1; anti-human 17-1A antibody(3622W94); anti-human colorectal tumor antibody (A33); anti-humanmelanoma antibody R24 directed against GD3 ganglioside; anti-humansquamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA)antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolym(Lym-1); anti-CCR5 (PRO 140); ABT-325; ABT-308; ABT-147; anti-beta7(etrolizumab); anti-HER3/EGFR DAF (DL11f); anti-interleukin 6 receptor(IL6R) such as tocilizumab (ACTEMRA®); and anti-Abeta (see WO2003/070760and WO2008/011348), etc.

In one embodiment the antibody which is formulated herein binds CD20 andis selected from: rituximab, ocrelizumab/humanized 2H7 (Genentech),ofatumumab (WO 04/035607, Genmab, Denmark), framework patched/humanizedIF5 (WO03/002607, Leung, S.), AME-133 (Applied Molecular Evolution), andhumanized A20 antibody (US 2003/0219433, Immunomedics).

In one embodiment the antibody which is formulated binds HER2 and istrastuzumab or pertuzumab.

In one embodiment the antibody which is formulated binds VEGF and isbevacizumab.

In one embodiment the antibody that is formulated herein is a humanizedantibody.

In one embodiment the antibody that is formulated is a recombinantantibody.

In one embodiment the antibody that is formulated has been expressed bya recombinant Chinese Hamster Ovary (CHO) cell.

In one embodiment the antibody that is formulated is a full lengthantibody.

In one embodiment the antibody that is formulated is a full length humanIgG1 antibody.

In one embodiment the antibody that is formulated is a full lengthhumanized IgG1 antibody.

In one embodiment the antibody that is formulated is a full lengthrecombinant humanized IgG1 antibody.

In one embodiment the antibody that is formulated is a full lengthhumanized IgG1 antibody that has been expressed by a recombinant ChineseHamster Ovary (CHO) cell.

In one embodiment the antibody that is formulated binds an antigenselected from: CD20 (e.g. rituximab), HER2 (e.g. trastuzumab), VEGF(bevacizumab), IL6R (tocilizumab), beta7 (etrolizumab). Abeta, HER3 andEGFR (DL11f), and M1′ (47H4v5).

In one embodiment the antibody formulated is rituximab.

In one embodiment the antibody formulated is trastuzumab.

In one embodiment the antibody formulated is bevacizumab.

III. The Pre-Spray Dried Preparation

A preparation of the monoclonal antibody is generally prepared which isto be subjected to spray drying, the so-called “pre-spray driedpreparation” herein.

In one embodiment, the pre-spray dried preparation comprises amonoclonal antibody preparation which has been subjected to one or moreprior purification steps, such as affinity chromatography (e.g. proteinA chromatography), hydrophobic interaction chromatography, ion exchangechromatography (anion and/or cation exchange chromatography), virusfiltration, etc. Thus, the antibody preparation may be purified,essentially pure, and/or essentially homogeneous.

In one embodiment, the monoclonal antibody in the pre-spray driedpreparation is concentrated. Exemplary methods for concentrating theantibody include filtration (such as tangential flow filtration orultrafiltration), dialysis etc.

The pre-spray dried preparation may be liquid or frozen.

The pH of the pre-spray dried preparation is optionally adjusted by abuffer. The buffer may for example have a pH from about 4 to about 8,e.g. from about 5 to 7, for example 5.8 to 6.2, and, in one embodiment,is approximately 6.0. A histidine buffer is an exemplified embodimentherein. The concentration of the buffer is dictated, at least in part,by the desired pH. Exemplary concentrations for the buffer are fromabout 1 mM to about 200 mM, or from about 10 mM to about 40 mM.

The pre-spray dried preparation optionally also comprises one or morestabilizers which prevent denaturation and/or aggregation of theantibody during the spray drying process. Examples of such stabilizersinclude saccharides (e.g. sucrose or trehalose) and/or surfactants (e.g.polysorbate 20 or polysorbate 80) and/or amino acids (e.g. histidine,arginine, glycine, and/or alanine). The stabilizers are generally addedin amount(s) which protect and/or stabilize the monoclonal antibody atthe lowest amount of stabilizer possible, to avoid increasing theviscosity of the final formulation.

With respect to saccharide stabilizers, such as disaccharides (e.g.trehalose or sucrose), the molar ratio of saccharide:monoclonal antibody(or disaccharide:monoclonal antibody) is optionally from about 50 toabout 400:1, e.g. from about 100 to about 250:1. Stated differently,exemplary saccharide concentrations in the pre-spray dried preparationare, for example, from about 10 mM to about IM, for example from about50 mM to about 300 mM.

With respect to surfactant (if included in the pre-spray dryingformulation), polysorbate 20 or polysorbate 80 are examples ofsurfactants that can be included. The surfactant is generally includedin an amount which reduces or prevents denaturation and/or aggregationof the monoclonal antibody during the spray drying process. Thesurfactant (e.g. polysorbate 20 or polysorbate 80) concentration isoptionally from about 0.0001% to about 1.0%, for example from about0.01% to about 0.1%.

The pre-spray dried preparation may be subjected to spray dryingprocedures such as those described in the following section.

IV. Spray Drying the Preparation

Spray drying herein is distinct from freeze drying commonly used toprepare monoclonal antibody formulations insofar as it is performed attemperatures above ambient temperature. Spray drying temperatures arecommonly expressed as “air inlet” and “air outlet” temperatures. In oneembodiment, the spray drying is performed at an air inlet temperaturefrom about 100° C. to about 220° C. (for example from about 120° C. toabout 160° C.) and an air outlet temperature from about 50° C. to about100° C. (for example from about 60° C. to about 80° C.).

The spray drying process generally comprises: atomization of the liquidfeed; drying of the droplets; and separation or recovery of the driedproduct.

Embodiments of atomizers herein include: rotary atomizers, pneumaticnozzle atomizers, ultrasonic nozzle atomizers, sonic nozzles, etc.

The contact between the liquid feed and the drying air can occur in twodifferent modes. In a co-current system, drying air and particles(droplets) move through the drying chamber in the same direction. Whendrying air and droplets move in an opposite direction, this is called acounter-current mode. Particles produced in counter-current mode usuallyshow a higher temperature than the exhausting air. The exhausted airitself can leave the system or can be recirculated. By choosing from thevarious spray dryer designs (size, atomizer, aseptic conditions, etc.)and adjusting the different process parameters (drying air flow, dryingair temperature, etc.), the final powder properties like particle size,shape and structure or even sterility can be modified. If the resultingmoisture of the recovered powder is not sufficiently low, post-treatmentmight be required, e.g., in the form of fluid bed dryers and coolers,contact dryers or even microwave dryers.

When the liquid feed is atomized, its surface to mass ratio isincreased, the heat transfer between the air and the droplets isaccelerated, and droplets can dry relatively rapidly. Two convectionprocesses may be involved: heat transfer (air to droplet) and masstransfer of moisture (droplet to air). In the latter, moisture permeatesthrough the boundary layer that surrounds each droplet. Transfer ratesmay be influenced by temperature, humidity, transport properties of thesurrounding air, droplet diameter and relative velocity between dropletand air.

The last step of a spray drying process is typically the separation ofthe powder from the air/gas and the removal of the dried product. Insome embodiments, this step is as effective as possible to obtain highpowder yields and to prevent air pollution through powder emission tothe atmosphere. To this end, various methods are available such ascyclones, bag filters, electrostatic precipitators, high pressure gas,electrostatic charge and combinations thereof.

The spray drying process produces particles comprising the monoclonalantibody.

In one embodiment, the characteristics of the spray dried powdercomprise any one or more or the following:

-   -   (a) average particle size: from about 2 microns to about 30        microns; e.g. from about 2 microns to about 10 microns;    -   (b) particle morphology: predominantly spherical particles, some        dimples or holes in particles, “dry raisin” shape;    -   (c) water content: less than about 10%, for example less than        about 5%, e.g., where water content is measured by a chemical        titration method (e.g. Karl Fischer method) or a weight-loss        method (high-temperature heating); and    -   (d) stability: e.g., assessed by suspending the particles in a        vehicle and evaluating physical stability and/or chemical        stability and/or biological activity of the suspension        preparation. In one embodiment, the percentage monomer of such        preparation is 95% to 100%, e.g. as evaluated by size exclusion        chromatography (SEC).

V. The Suspension Formulation

The spray dried monoclonal antibody particles prepared as described inthe preceding section are combined with a non-aqueous suspension vehicleto generate the suspension formulation. This formulation is suitable foradministration to a subject. Generally, the suspension formulation willnot be subjected to either prior, or subsequent, lyophilization orcrystallization. In one embodiment, a subcutaneous administration device(e.g. a pre-filled syringe) is filled with the suspension formulationand used for administering the formulation (see below for more detaileddisclosure regarding devices and methods of treatment).

The invention also provides a method of making a suspension formulationcomprising suspending the spray dried monoclonal antibody in anon-aqueous suspension vehicle.

In one embodiment the antibody concentration in the suspensionformulation is about 200 mg/mL or more.

In one embodiment the antibody concentration in the suspensionformulation is from about 200 mg/mL to about 500 mg/mL.

In one embodiment the antibody concentration in the suspensionformulation is from about 250 mg/mL to about 400 mg/mL.

In one embodiment the antibody concentration in the suspensionformulation is from about about 250 mg/mL to about 350 mg/mL.

The non-aqueous suspension vehicle preferably has a viscosity at 25° C.,which is less than about 20 centipoise, for example, less than about 10centipoise, and optionally less than than about 5 centipoise.

According to one embodiment of the invention, the viscosity of thesuspension formulation is from about 5 to about 100 centipoise, forinstance, from about 10 to about 70 centipoise at 25° C. In oneembodiment, viscosity of the suspension formulation is measured using acone and plate rheometer (e.g. a AR-G2 TA Instrument rheometer).

In one embodiment, the average particle size in the suspensionformulation is from about 2 microns to about 30 microns, for examplefrom about 5 microns to about 10 microns.

In one embodiment, the suspension formulation has an injection glideforce of less than about 20 newton, for example less than about about 15newton. Such injection glide force may be determined as a function ofmonoclonal antibody concentration by injecting 1-mL suspension using a1-mL long syringe through a 27-gauge TW staked needle in 10 seconds.

In one embodiment the non-aqueous suspension vehicle is selected from:propylene glycol dicarprylate/dicaprate, benzyl benzoate, ethyl lactate,or mixtures of two or three thereof.

In one embodiment the non-aqueous suspension vehicle comprises ethyllactate.

In one embodiment, the non-aqueous suspension vehicle comprises amixture of at least two non-aqueous suspension vehicles: Vehicle A plusVehicle B, wherein the viscosity of Vehicle A is less than that ofVehicle B, but the monoclonal antibody stability in Vehicle B is greaterthan that in Vehicle A. An embodiment of such mixture is exemplified bythe mixture of ethyl lactate and propylene glycol dicarprylate/dicaprate(for example).

In one aspect, the suspension formulation comprises a spray dried fulllength human IgG1 monoclonal antibody at a concentration from about 200mg/mL to about 400 mg/mL suspended in a non-aqueous suspension vehiclewith a viscosity less than about 20 centipoise, wherein the formulationhas an average particle size from about 2 microns to about 10 microns,and injection glide force less than about 15 newton.

The suspension formulation optionally further comprises one or moreexcipients or stabilizers. Examples of such stabilizers includesaccharides (e.g. sucrose or trehalose) and/or surfactants (e.g.polysorbate 20 or polysorbate 80) and/or amino acids (e.g. histidine,arginine, glycine, and/or alanine). The stabilizers are generallypresent in an amount which protects and/or stabilizes the monoclonalantibody at the lowest amount of stabilizer possible, to avoidincreasing the viscosity of the suspension formulation. In oneembodiment, the stabilizers are present in the suspension formulation asa result of having been added to the pre-spray dried preparation, and/orhave been added to the suspension formulation, as desired.

With respect to saccharide stabilizers, such as disaccharides (e.g.trehalose or sucrose), the molar ratio of saccharide:monoclonal antibody(or disaccharide:monoclonal antibody) in the suspension formulation isoptionally from about 50 to about 400:1, e.g. from about 100 to about250:1. Stated differently, exemplary saccharide concentrations in thesuspension formulation are from about 10 mM to about 1 M, for examplefrom about 50 mM to about 300 mM.

With respect to surfactant (if included in the pre-spray driedpreparation), polysorbate 20 or polysorbate 80 are examples ofsurfactants which can be present in the suspension formulation. Thesurfactant (e.g. polysorbate 20 or polysorbate 80) concentration isoptionally from about 0.0001% to about 1.0%, for example from about0.01% to about 0.1%.

The suspension formulation is generally sterile, and this can beachieved according to the procedures known to the skilled person forgenerating sterile pharmaceutical formulations suitable foradministration to human subjects, including filtration through sterilefiltration membranes, prior to, or following, preparation of thesuspension formulation.

Moreover, the formulation is desirably one which has been demonstratedto be stable upon storage. Various stability assays are available to theskilled practitioner for confirming the stability of the formulation.Stability can be tested by evaluating physical stability, chemicalstability, and/or biological activity of the antibody in the suspensionformulation around the time of formulation as well as following storageat different temperatures and time-points. In one embodiment, monoclonalantibody stability is assessed by size distribution (percentage monomer,aggregation, and/or fragmentation) before and after spray drying (e.g.before and after spray drying over 3-month storage under the acceleratedtemperature of 40° C.). In one embodiment, size distribution is assessedusing using size exclusion chromatography-high performance liquidchromatography (SEC-HPLC). In one embodiment, the percentage monomerloss in the suspension formulation (as measured by SEC-HPLC) over 3months is less than about 10%, for example less than about 5%.

In one embodiment, the invention provides a method of making apharmaceutical formulation comprising preparing the suspensionformulation as described herein, and evaluating any one or more of thefollowing properties of the formulation:

-   -   (a) physical stability, chemical stability, and/or biological        activity of the monoclonal antibody in the suspension (e.g.        measuring percentage monomer using size exclusion        chromatography);    -   (b) viscosity of the suspension formulation;    -   (c) injectability or injection glide force of the suspension        formulation;    -   (d) surface energy analysis (SEA) or heat of sorption, e.g. by        inverse gas chromatography (IGC) to evaluate particle-suspension        vehicle interaction;    -   (e) particle size (e.g. average and/or peak particle size, e.g.        by laser diffraction analyzer); and/or    -   (e) suspension physical stability (settling, homogeneity over        time, particle sedimentation rate, etc).

Further detail of exemplary assays for these properties is provided inthe example below.

One or more additional other pharmaceutically acceptable carriers,excipients or stabilizers such as those described in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may beincluded in the formulation provided that they do not adversely affectthe desired characteristics of the formulation. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and include; additional buffering agents;co-solvents; antioxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; preservatives;and/or salt-forming counterions such as sodium.

VI. Medicaments and Treatments Using the Suspension Formulation

In one embodiment, the invention provides a method of treating a diseaseor disorder in a subject comprising administering the suspensionformulation described herein to a subject in an amount effective totreat the disease or disorder.

Thus, the invention provides: the suspension formulation as describedherein for treating a patient in need of treatment with the monoclonalantibody in the suspension formulation; and use of the suspensionformulation in the preparation of a medicament for treating a patient inneed of treatment with the monoclonal antibody in the suspensionformulation. In an alternative embodiment, the invention provides: theformulation as described herein for treating a disease or disorder in apatient; and use of the formulation in the preparation of a medicamentfor treating a disease or disorder in a patient.

In addition, the invention provides a method of treating a patientcomprising administering the formulation described herein to a patientin order to treat a disease or disorder in the subject. Preferably theformulation is administered subcutaneously to the subject or patient. Inone embodiment, the formulation is administered by a pre-filled syringecontaining the formulation therein.

Where the antibody in the formulation binds to HER2, the suspensionformulation is preferably used to treat cancer. The cancer willgenerally comprise HER2-expressing cells, such that the HER2 antibodyherein is able to bind to the cancer cells. Thus, the invention in thisembodiment concerns a method for treating HER2-expressing cancer in asubject, comprising administering the HER2 antibody pharmaceuticalformulation to the subject in an amount effective to treat the cancer.Exemplary cancers to be treated herein with a HER2 antibody (e.g.trastuzumab or pertuzumab) are HER2-positive breast cancer or gastriccancer.

Where the antibody in the formulation binds to a B-cell surface markersuch as CD20, the formulation may be used to treat a B-cell malignancy,such as NHL or CLL, or an autoimmune disease (e.g. rheumatoid arthritisor vasculitis).

Where the antibody in the formulation binds VEGF (e.g. bevacizumab), theformulation may be used to inhibit angiognesis, treat cancer (such ascolorectal, non-small cell lung (NSCL), glioblastoma, breast cancer, andrenal cell carcinoma), or treat age-related macular degeneration (AMD)or macular edema.

Where the indication is cancer, the patient may be treated with acombination of the suspension formulation, and a chemotherapeutic agent.The combined administration includes coadministration or concurrentadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinthere is a time period when both (or all) active agents simultaneouslyexert their biological activities. Thus, the chemotherapeutic agent maybe administered prior to, or following, administration of thecomposition. In this embodiment, the timing between at least oneadministration of the chemotherapeutic agent and at least oneadministration of the formulation is preferably approximately 1 month orless, and most preferably approximately 2 weeks or less. Alternatively,the chemotherapeutic agent and the formulation are administeredconcurrently to the patient, in a single formulation or separateformulations.

Treatment with the suspension formulation will result in an improvementin the signs or symptoms of the disease or disorder. Moreover, treatmentwith the combination of the chemotherapeutic agent and the antibodyformulation may result in a synergistic, or greater than additive,therapeutic benefit to the patient.

The formulation is administered to a human patient in accord with knownmethods, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, or intrathecal administration.

Intramuscular or subcutaneous administration of antibody composition ispreferred, with subcutaneous administration being most preferred.

For subcutaneous delivery, the formulation may be administered viasyringe (e.g. pre-filled syringe); autoinjector; injection device (e.g.the INJECT-EASE™ and GENJECT™ device); injector pen (such as theGENPEN™); or other device suitable for administering a suspensionformulation subcutaneously. The preferred device herein is a pre-filledsyringe.

For the prevention or treatment of disease, the appropriate dosage ofthe monoclonal antibody will depend on the type of disease to betreated, as defined above, the severity and course of the disease,whether the monoclonal antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the monoclonal antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 50 mg/kg (e.g. 0.1-20mg/kg) of antibody is an initial candidate dosage for administration tothe patient, whether, for example, by one or more separateadministrations, or by continuous infusion. The dosage of the antibodywill generally be from about 0.05 mg/kg to about 10 mg/kg. If achemotherapeutic agent is administered, it is usually administered atdosages known therefor, or optionally lowered due to combined action ofthe drugs or negative side effects attributable to administration of thechemotherapeutic agent. Preparation and dosing schedules for suchchemotherapeutic agents may be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992).

VII. Articles of Manufacture

The invention herein also concerns a device with the suspensionformulation therein. Preferably the device is a subcutaneousadministration device, such as a pre-filled syringe.

In a related aspect, the invention provides a method of making anarticle of manufacture comprising filling a container with thesuspension formulation.

Embodiments of the container in the article of manufacture include:syringes (such as pre-filled syringe), autoinjectors, bottles, vials(e.g. dual chamber vials), and test tubes, etc. The container holds thesuspension formulation and the label on, or associated with, thecontainer may indicate directions for use. The article of manufacturemay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use as noted in theprevious section.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

EXAMPLES

Developing high-concentration monoclonal antibody liquid formulations(≥200 mg/mL) for subcutaneous (SC) administration is often challengingwith increased viscosity that makes injection difficult. Thisinvestigation was intended to overcome this obstacle using a non-aqueouspowder suspension approach. Three human IgG1 monoclonal antibodies werespray dried and suspended in a suspension vehicle at differentmonoclonal antibody concentrations. Propylene glycoldicaprylate/dicaprate, benzyl benzoate, and ethyl lactate were employedas model suspension vehicles. Suspensions were characterized forviscosity, particle size, and syringeability. Physical stability of thesuspension was visually inspected. The suspensions in generaloutperformed the liquid solutions in terms of injectability despitehigher viscosity at the same monoclonal antibody concentrations. Powderformulations and powder properties appeared to have little effect onsuspension viscosity or injectability. Among the three suspensionvehicles, ethyl lactate suspensions had the lowest viscosity, below 20centipoise, and lowest syringe injection glide force, below 15 newton,at monoclonal antibody concentration as high as 333 mg/mL (total powderconcentration at 500 mg/mL). Inverse gas chromatography (IGC) analysisof the suspension supported the conclusion that the suspension vehiclewas the most important factor impacting suspension performance. Ethyllactate rendered greater heat of sorption than other suspensionvehicles. Without being bound by any one theory, this indicates thatstrong particle-suspension vehicle interaction may reduceparticle-particle self association, leading to low suspension viscosityand glide force. Ethyl lactate suspensions, however, lacked the physicalsuspension stability exhibited by propylene glycol dicaprylate/dicaprateand benzyl benzoate. Specific mixtures of ethyl lactate and propyleneglycol dicaprylate/dicaprate improved the overall suspension performancein high monoclonal antibody concentration suspensions.

Amongst other things, these examples demonstrated the viability of highmonoclonal antibody concentration (>300 mg/mL) in suspensionformulations for SC administration.

Materials and Methods

Three recombinant chimeric/humanized monoclonal antibodies of the humanIgG1 subclass bevacizumab, trastuzumab and rituximab were manufacturedby Genentech (South San Francisco, Calif.). These antibodies wereexpressed by Chinese hamster ovary (CHO) cell lines. All antibody drugsubstance liquid solutions were concentrated to 100 mg/mL using atangential-flow filtration unit (PELLICON3® 10 kD, Millipore, Billerica,Mass.) and formulated with trehalose dihydrate. All bulks were bufferedto a pH of ˜6.0. For antibody powder suspension preparation, propyleneglycol dicaprylate/dicaprate (Batch #091125, SASOL, Hamburg, Germany),benzyl benzoate (Cat # B9550, Sigma-Aldrich, St Louis, Mo.), and ethyllactate (Lot #BCBC7752, Sigma-Aldrich, St. Louis, Mo.) were used assuspension vehicles.

Spray Drying

Two types of spray dryers were used in this study, a pilot-scale unit(MS-35, SPX Flow Technology Systems, Inc., Elkridge, Md.) and abench-top unit (B-191, Buchi Corp., New Castle, Del.). MS-35 isapproximately 2-fold larger capacity than B-191, i.e., 2.5 vs. 1.6kg/hour of the maximum water evaporation rate and 35 vs. 20 kg/hourmaximum compressed air consumption rate. The pilot-scale unit wasconstructed mostly of stainless steel with heat insulation (dryingchamber, cyclone, etc.) while the bench-top unit was made of glass. Thepilot scale unit was equipped with a high-efficiency cyclone. Tocalculate the yield of powder collection, only the powder collected inthe receiver was considered for the pilot-scale unit, and the powdercollected on the cyclone and the receiver lid was included for thebench-top unit. The spray drying conditions and the characteristics drypowders produced using both spray dryers are listed in Table 2.

TABLE 2 Spray-drying conditions in two types of spray dryers andcharacterization results of three antibodies formulated with trehaloseat 1:2 antibody:trehalose weight ratio Monoclonal Antibody TypeBevacizumab Trastuzumab Rituximab Spray Dryer Pilot Bench-top PilotBench-top Pilot Bench-top Drying Inlet Temp. 182 134 182 138 182 136Condition (° C.) Outlet Temp. 87 88 87 89 87 88 (° C.) Liq Feed Rate 123 12 3 13 3 (mL/min) Liq Vol Dried 250 50 250 50 250 50 (mL) Yield (%)99 60 100 65 98 59 Particle Size (D₅₀) (μm) 9.6 2.5 8.8 2.8 10.6 5.1Water Content (%) 4.0 7.6 4.7 6.9 5.0 8.8

Freeze Drying

Monoclonal antibody solutions were also freeze-dried to compare thedry-state stability with spray dried samples. Liquid formulations werealiquoted in 1 mL into 2 cc glass vials placed with butyl stoppers, thenplaced on pre-chilled shelves at −50° C. in a lyophilizer (Model #LYOMAX2®, BOC Edward, Tewksbury, Mass.). The samples were dried bylowering the pressure to 100 mTorr and increasing the shelf temperatureto −25° C. during the primary drying, followed by the secondary dryingat 35′C. The total lyophilization cycle time was approximately 60 hours.

Particle Size Analysis

The particle size distribution was measured using a laser diffractionanalyzer (LA-950, Horiba Instruments, Kyoto, Japan). The LA-950 consistsof two light sources (blue LAD, red laser), a sample handling system tocontrol the interaction of particles and incident light, and an array ofhigh quality photodiodes to detect the scattered light over a wide rangeof angles. The scattered light collected on the detectors was used tocalculate the particle size distribution of the sample analyzed usingthe Mie Theory. For spray dried samples, several milligrams of the drypowders were dispersed in 50 mL of isopropyl alcohol in the MiniFlowcell attached on LA-950 and sonicated using the sonicator also attachedon IA-950 for about one minutes prior to analysis. For particlessuspended in vehicles were diluted with each vehicle in FractionCell andmix with a stirrer attached on LA-950 prior to analysis.

Density Analysis

The density of the powder was determined by mixing 500 mg of powder in 4mL of propylene glycol dicaprylate/dicaprate oil in a volumetriccylinder and measuring the displaced oil volume as the powder volume.Powder density can be calculated using powder weight and volume.

Scanning Electron Microscopy

Surface morphology of spray dried samples was examined using anenvironmental scanning electron microscope (XL30, FEL, Hillsboro,Oreg.). Each sample was mounted on aluminum stubs and sputter coatedwith 10 nm layer of AuPd, and scanned at a voltage of 2 kV, and thephotographs were taken at magnifications of 1000 and 2000.

Water Content Analysis

Residual moisture in spray dried samples were determined usingvolumetric Karl Fischer titration analyzer (DL31, Mettler-Toledo).Approximately 100 mg of each sample was injected into the titration cellthat contained anhydrous methanol. Hydranal composite 2 volumetricreagent (Cat #34696, Hiedel-deHaen, Heidelberg, Germany) was used as atitrant.

Size Exclusion Chromatography

The quantitation of size variants was determined by si e exclusionchromatography. This analysis utilized a G3000SW_(XL) column, 7.8 mmID×30 cm, 5 μm (TOSOH BioScience) run on an HPLC system (1100, Agilent).The mobile phases are 0.2 M potassium phosphate and 0.25 M potassiumchloride at pH 6.2 for bevacizumab, 0.1 M potassium phosphate at pH 6.8for trastuzumab, and 0.2 M potassium phosphate and 0.25 M potassiumchloride at pH 7.0 for rituximab. The chromatography was runisocractically at a flow rate of 0.5 mL/min for 30 minutes. The columntemperature was maintained at ambient for bevacizumab and rituximab, and30° C. for trastuzumab, and the eluent absorbance was monitored at 280nm. Each monoclonal antibody was diluted with its respective formulationbuffer to 25 mg/mL for bevacizumab and 10 mg/mL for both trastuzumab andrituximab. Their injection volume is 10 μL for bevacizumab and for 20 μLfor both trastuzumab and rituximab.

Monoclonal Antibody Physical Stability in Spray dried and Freeze-DriedPowder Formulations

Spray dried and freeze-dried powder samples were aliquotted into 2 ccglass vial, approximately 25 monoclonal antibody. Each vial was sealedwith a rubber stopper and FLIP-OFF® cap and stored at 40° C. for up to 3months. At the stability time points of time zero (immediately afterdrying), 1, 2, 3 months, each dry sample was reconstituted with 1 mL, ofpurified water, and the antibody physical stability was determined byprotein size distribution (% monomer, aggregation, and fragmentation)using SEC-HPLC.

Preparation of Suspension Formulations

The powder was weighed onto a 2-mL vial. Based on the powder densitydetermined, the appropriate amount of suspension vehicle was added toprepare the powder concentration in the unit of mg of powder in 1 mL ofsuspension volume. Samples were then homogenized for 2 minutes at 7500rpm using a 0.5-cm tip probe on a Tempest Virtishear homogenizer (ViritsCorp, Gardiner, N.Y.).

Viscosity Measurement

The viscosity of solution and suspension samples was measured using acone and plate rheometer (AR-G2 TA Instrument, New Castle, Del.). Eachsample was loaded onto the lower measuring plate and allowed to come tothermal equilibrium at 25° C. A solvent trap equipped on AR-G2 was usedto prevent solution evaporation during the measurement. The sampleviscosity was measured every 10 seconds for 2 minutes using a cone witha 20 mm diameter and 1 degree angle at shear rate of 1000 per second.

Syringe Glide Force Measurement

One mL of suspension was drawn into a 1.0 mL-Long 27G TW ½″ stakedneedle syringe (BD, Franklin Lakes, N.J.) sealed with a plunger stopper(W4023/FLT, West Pharmaceutical, Lionville, Pa.). The internal barrel ofthe syringe was coated with 0.5 mg silicone oil (Dow 360 Medical Fluid,1.000 cSt). A Material Testing System (Model 5542, Instron, Grove City,Pa.) with a load cell was used to apply a steady compression rate of 190mm/min. The gliding force profile was analyzed and established as afunction of the distance of the plunger rod travelling inside thesyringe barrel.

Inverse Gas Chromatography (IGC)

IGC experiment was performed using a Surface Energy Analysis (SEA)System (MSM-iGC 2000, Surface Measurement Services Ltd, Allentown, Pa.).Approximately 200 mg of powder sample was packed into individualsilanised glass columns and both ends of columns were sealed usingsilanised glass wool to prevent sample movement. The specific surfaceareas of the powder samples were determined by measuring the Octaneadsorption isotherms at 30° C. and 0% RH from the IGC SEA. The BETspecific surface areas of the samples were subsequently calculated fromtheir corresponding octane isotherms, within the partial pressure range(10% to 35% P/P₀). Decane, nonane, octane and heptane were used asalkane probes for dispersive surface energy determination. Specificacid-based Gibbs free energy was also measured using acetone,acetonitrile, ethanol and ethyl acetate. For heat of sorptionmeasurement, the suspension vehicles were used as the gaseous probes.All samples were pre-conditioned in-situ with a carrier gas of helium at30° C. for 2 hours, and all the measurements were conducted at 30° C.with a carrier gas flow rate of 10 cm/sec.

Results and Discussion Spray Dried Antibody/Trehalose Powders

Three types of monoclonal antibodies were formulated in liquid solutionscontaining trehalose, serving as a carbohydrate stabilizer to monoclonalantibody, at the weight ratio of 1:2 of trehalose:antibody prior tospray drying. This low weight ratio is equivalent to approximately 220:1molar ratio was used for the purpose of minimizing its volumecontribution, which was below the minimum molar ratio of 300:1 commonlyused for sugar to stabilize proteins as a lyoprotectant (Shire et al., JPharm Sci 93:1390-1402 (2004)). Note that a 400 mg powder/mL suspensionrepresents a 270 mg antibody/mL concentration even at the 1:2 weightratio of trehalose:antibody, which was at the low limit of the targetantibody concentration for this study.

Three monoclonal antibodies formulated at 100 mg/mL with 50 mg/mLtrehalose, were spray dried using a bench-top spray dryer (B-191) and apilot-scale spray dryer (MS-35). Spray-drying conditions and powdercharacterization results are summarized in Table 2. Comparable outlettemperatures of 87-89° C. were employed for all samples because outlettemperature was considered the key parameter dictating the spray-dryingcapability (Maa et al., Pharm Dev Technol 2:213-223 (1997); Lee G. SprayDrying of Proteins, in “Rational Protein Formulation: Theory andPractice” (Eds. Carpenter J, Manning M), Pharmaceutical BiotechnologySeries (Ed. Borchardt R). Plenum Press, pp. 135-158 (2002); Maury et al.Eur. J. Pharm. Biopharm. 59:566-573 (2005); Maa et al. Biotech. Bioeng.60:301-309 (1998); and Maa et al. J. Pharm. Sci. 87:152-159 (1998)). Thepilot-scale spray dryer demonstrated better performance in powdercollection yield (>96%) and water content of 4-5%, while the samplesdried by the bench-top spray dryer had 60% yield and 7-9% water content.The pilot-scale dryer was also capable of producing larger particles of8-11 rpm (D₅₀) whereas the bench-top dryer produced 2-5 μm (D₅₀)particles. The advantages of the pilot-scale dryer can be attributed toefficient energy use and greater powder collection efficiency. Particleshape and morphology for all antibodies was generally spherical withdimples, which were antibody dependent. The type of the spray dryer didnot affect particle morphology. Overall, dryer performance and theantibody type resulted in some degree of variations in particleproperties. Although these variations are not dramatic, they allowed usto evaluate their effect on suspension performance.

Antibody Physical Stability in Spray Dried and Freeze-Dried PowderFormulations

A general concern about spray drying of biologics was high temperaturestress, particularly for the pilot dryer which had higher inlettemperature of >180° C. Antibody physical stability of the dry sampleswas determined upon reconstitution with purified water by protein sizedistribution (% monomer, aggregation and fragmentation) using SEC-HPLCbefore and after spray drying over 3-month storage under the acceleratedtemperature of 40° C. (FIG. 1). Despite the high drying temperature usedin the pilot-scale spray dryer, the impact of the drying process on (%)monomer was minimal. The antibody physical stability for spray driedbevacizumab and trastuzumab was compared to the freeze-driedcounterparts by monitoring the change in (%) monomer at 40° C. over 3months. The (%) monomer for all samples decreased at the acceleratedcondition mainly due to aggregation, which is not surprising given thesub-optimal amount of trehalose to protect antibody in the formulation.However, the spray dried samples had greater antibody physical stabilitythan the freeze-dried samples. The (%) monomer of spray driedtrastuzumab and bevacizumab decreased by ˜2% and ˜4% respectively,whereas both freeze-dried antibodies suffered a greater (%) monomer lossof −6.5% over 3 months, despite their lower water content of −0.8%.Thus, spray drying is a viable approach, from the process and stabilityperspective, in making antibody powders for suspension formulationdevelopment.

Selection of Suspension Vehicles

The primary criterion for the selection of the suspension vehicle waslow viscosity, preferably <10 Cp, as suspension vehicle viscosity wouldcontribute to suspension viscosity in a linear fashion based onEinstein's Equation for the viscosity of solutions (Einstein, A.,Annalen der Physik 34:591-92 (1911)).

η=η_(o)(1+2.5φ)  (Equation 2)

Where η is the suspension viscosity. η_(o) the viscosity of puresuspension vehicle, and φ the volume fraction of the solute.

The three suspension vehicles selected for this study, propylene glycoldicaprylate/dicaprate, benzyl benzoate, and ethyl lactate, met thiscriterion (Table 3). MIGLYOL 840® is propylene glycol diesters ofcaprylic and capric acids from the MIGLYOL® neutral oil family. MIGLYOL810® and MIGLYOL 812® have been approved for intravenous andintramuscular injections but they are viscous, >30 cp at ambienttemperature. Propylene glycol dicaprylate/dicaprate, the least viscousin the family (˜9 cp), has been used for transdermal applications(Mahjour et al., Intl J Pharm 95:161-169 (1999); Seniro, W., Intl JToxicol 18:35-52 (1999)). Benzyl benzoate is similar to propylene glycoldicaprylate/dicaprate in viscosity, ˜9 cp, and has often been used as apreservative in liquid injectables at <10% concentration. Ethyl lactatehas been used commonly in pharmaceutical preparations, food additives,and fragrances due to its relatively low toxicity. Although ethyllactate has not yet been parenterally approved, it had low toxicity inmice for intramuscular and intravenous injection (Spiegel andNoseworthy, J Pharmn Sci 52:917-927 (1963); Mottu et al., PDA J. Pharm.Sci. Technol. 54:456-469 (2000)). Ethyl lactate has a water-likeviscosity, ˜2 cp.

TABLE 3 Structure, viscosity, and pharmaceutical application informationof three model suspension vehicles tested in this study Miglyol 840Ethyl Lactate Benzyl Benzoate Structure

Viscosity (cp) at 20° C. 9 2 9 Pharmaceutical Not currently approved forUsed as flavor enhancer for Used as a preservative in Applicationsparenteral use, but some animal oral dose medications. Not liquid dosageform for tox studies have been approved for parenteral use parenteraladministration in, conducted for skin delivery but acute toxcity in miceby quantities less than 10% SC and IV are available

Effect of Antibody Type and Powder Properties on Suspension Viscosity

All antibodies dried by both bench-top and pilot-scale spray dryers(Table 2) were suspended in propylene glycol dicaprylate/dicaprate.Suspension viscosity was measured as a function of antibodyconcentration, and compared to the antibody liquid solutions (FIG. 2).Suspension viscosity for all antibodies was similar in the range ofantibody concentration tested, suggesting that variations in antibodytypes and powder properties (particle size, morphology, and moisturecontent) had little effect on suspension viscosity. Suspension viscosityincreased with increasing antibody concentration in an exponentialmanner, which can be expressed as:

η_(Miglyol 840)=8.24e ^(0.0088(powder conc))  (Equation 3)

Certainly, it is very different from the Einstein equation (Equation 2)which is primarily for dilute suspensions. Equation 4, a modifiedversion of Equation 2, took the interactions of more concentratedsuspensions into consideration (Kunitz, M., J. General Physiology pages715-725 (July 1926)), however, it still significantly underestimated theempirical data (see the dash line in FIG. 2).

η/η_(o)=(1+0.5φ)/(1−φ)⁴  (Equation 4)

It was interesting to find that suspension viscosity was actually higherthan the viscosity of the corresponding antibody liquid solution at thesame antibody concentration. No difference in suspension viscosity wasobserved among the antibodies, although the type of antibody didsignificantly affect liquid viscosity.

Surface Energies of Spray Dried Powders by IGC

Kanai and co-workers (Kanai et al., J. Pharm. Sci. 97:4219-4227 (2005))found reversible self-association as the result of Fab-Fab interactionsin their viscosity study tested with two antibodies made of the sameconstruct with different amino acid sequences in the complementaritydetermining region (CDR) region in aqueous solutions. Such viscositydifferences due to the antibody types in powder suspensions innon-aqueous vehicles were not observed (FIG. 2). This observation couldbe interpreted from the perspective of particle surface energydistribution in the powder suspension. Particle surface energy, thecombination of polar and non-polar (dispersive) energy components, candictate the level of interactions with suspension vehicles andparticles. IGC is a common tool for surface energy measurement. Theparticle's dispersive surface energy using decane, nonane, octane andheptane as the probes, and also specific acid-base (polar) Gibbs freeenergy were measured using acetone, ethyl acetate, ethanol, andacetonitrile as the probes. Surface energy is a distribution in responseto particle size distribution of the powder sample but only surfaceenergies at the 50% values were reported in Table 4. The dispersivesurface energy, γ₅₀, was in a narrow range of 36 to 38 mJ/m² for allthree antibodies. The differences in specific acid-base Gibbs freeenergy, ΔG₅₀, of these antibodies in response to the four acid-baseprobes were also in a narrow range of 8 to 13 mJ/m². The comparablesurface energy distribution among the three antibody powders couldexplain similar particle-suspension vehicle and particle-particleinteractions, leading to their comparable suspension viscosity inpropylene glycol dicaprylate/dicaprate (FIG. 2).

TABLE 4 Dispersive surface energy (γ₅₀), specific acid-base Gibbs freeenergy (ΔG₅₀), and heat of sorption of spray dried monoclonal antibodypowders (all measured using IGC) Heat of Sorption γ₅₀ ΔG₅₀ ΔH_(sorption)Powder Suspension Vehicles (mJ/m²) (mJ/m²) (KJ/mole) Bevacizumab Decane,nonane, 37.5 octane and heptane Trastuzumab Decane, nonane, 36.8 octaneand heptane Rituximab Decane, nonane, 38.3 octane and heptaneBevacizurnab Acetone 8.4 Ethyl acetate 6.2 Ethanol 14.8 Acetonitrile12.9 Trastuzumab Acetone 8.2 Ethyl acetate 6.6 Ethanol 14.5 Acetonitrile12.7 Rituximab Acetone 8.4 Ethyl acetate 7.3 Ethanol 14.9 Acetonitrile12.8 Bevacizumab Propylene glycol 39.9 ± 0.5 dicaprylate/dicaprateBenzyl benzoate 36.5 ± 0.7 Ethyl lactate 51.5 ± 0.3 Rituximab Propyleneglycol 43.4 ± 0.5 dicaprylate/dicaprate Benzyl benzoate 42.8 ± 0.6 Ethyllactate 58.5 ± 0.4

Injectability of Suspensions in Three Vehicles

Injectability can be monitored by glide force measurement, which is aperformance indicator more relevant than viscosity measurement. Theglide force of the rituximab powder suspension in three vehicles wasdetermined as a function of antibody concentration by injecting 1-mLsuspension using a 1-mL long syringe through a 27-gauge TW staked needlein 10 seconds (FIG. 3). The glide force for all suspensions increasedwith antibody concentration, however, it was below 20 N even at 200mg/mL antibody concentration despite the high viscosity (FIG. 2). Thepredicted glide force for the antibody liquid solutions extracted fromFIG. 4 in Reference 3 was higher than the suspension glide force. Theglide force in ethyl lactate suspension was lowest among the threesuspension vehicles tested. The glide force of the ethyl lactatesuspension at 333 mg antibody/mL was equivalent to that in the other twosuspension vehicles at about half of the antibody concentration (167mg/mL), which was still below the target threshold of 15 newton, even athigh antibody concentration of 333 mg/mL. The reasons for theviscosity-glide force relationship discrepancy between the liquidsolution and the suspension are not clear.

Effect of Suspension Vehicle on Suspension Viscosity

Suspension viscosity was tested in three vehicles containing the spraydried rituximab powder (FIG. 4). The viscosity in ethyl lactate was thelowest among the three vehicles; the viscosity of the ethyl lactatesuspension at 333 mg antibody/mL was equivalent to that of thesuspension in propylene glycol dicaprylate/dicaprate and benzyl benzoateat about half of the antibody concentration (167 mg/mL).

Heat of Sorption by IGC and Particle Size

Heat of sorption (ΔH_(sorption)) is a direct measure of the strength ofthe interactions between a solid and gas molecules adsorbed on thesurface (Thielmann F., “Inverse gas chromatography: Characterization ofalumina and related surfaces,” In “Encyclopedia of Surface and ColloidScience Volume 4 (edit by P. Somasundaran) CRC Press, Boca Raton. Fla.,p3009-3031 (2006); Thielmann and Butler, “Heat of sorption onmicrocrystalline cellulose by pulse inverse gas chromatography atinfinite dilution,” Surface Measurement Services Application Note 203(http://www.thesorptionsolution.com/Information_Application_Notes_IGC.php#Aps)(2007)).

The IGC method was employed to measure the heat of sorption betweenspray dried particles and the suspension vehicles (Table 4). For bothbevacizumab and rituximab, ethyl lactate suspension had higher heat ofsorption than the other two suspension vehicles. Particle size of thesuspension particles was also compared among the three suspensions (FIG.5). The peak particle size (highest percentage) was 28, 25, and 7 mm forpropylene glycol dicaprylate/dicaprate, benzyl benzoate and ethyllactate, respectively. Both heat of sorption and particle size data showthat the higher heat of sorption in ethyl lactate suspensions indicatedhigher particle-suspension vehicle interaction than particle-particleinteraction and that the degree of particle self-association in ethyllactate was lower than that in propylene glycol dicaprylate/dicaprate orbenzyl benzoate.

Suspension Physical Stability

Despite low viscosity and glide force in the ethyl lactate suspension,it displayed a peculiar suspension physical stability as a function oftime. The powder in the ethyl lactate suspension settled to the bottomand floated to the surface of the suspension after 1-day ambient storage(FIG. 6A). Homogeneity of the ethyl lactate suspension could be restoredby vortexing (FIG. 6B). On the contrary, the suspension physicalstability in propylene glycol dicaprylate/dicaprate was much more stableand remained well suspended over two weeks (FIG. 6C).

According to the particle sedimentation rate determined by Stoke's Law(Eq. 4 below), the particles in ethyl lactate would settle approximately4.5 times faster than in propylene glycol dicaprylate/dicaprate, basedon the density and viscosity of ethyl lactate and propylene glycoldicaprylate/dicaprate, 1.03 g/cm³ and 0.92 g/cm³, and 2 cP and 9 cP,respectively. Thus, Stoke's Law alone couldn't fully explain theobservation of extremely fast settlement of particles in ethyl lactateas compared to propylene glycol dicaprylate/dicaprate, suggesting othermechanisms such as surface electrical charge (i.e., zeta potential) mayplay a role. However, the phenomenon of some of the particles floatingto the top of ethyl lactate surface is difficult to explain because thedensity of the spray dried particles is higher than ethyl lactate.

s=d(ρ_(s)−ρ₁)g/(18η)  (Equation 5)

where s is sedimentation rate, d diameter of the particle, ρ_(s) thedensity of the particle, ρ₁ the density of the suspension vehicle, gacceleration due to gravity, and η the viscosity of the suspensionvehicle.

Suspension Vehicle Mixture to Improve Suspension Performance

The mixtures of ethyl lactate and propylene glycol dicaprylate/dicapratewere used as suspension vehicles for testing rituximab suspensionphysical stability. Particle size was determined for these mixturesuspensions (FIG. 7A). The particle size decreased with decreasingpropylene glycol dicaprylate/dicaprate contribution in the mixture wherethe peak particle size was 28, 13, 11, 8 and 7 μm for propylene glycoldicaprylate/dicaprate:ethyl lactate mixture at 100:0, 75:25, 50:50,25:75, and 0:100, respectively. From the suspension physical stabilityperspective, the poor suspension stability of ethyl lactate was improvedby mixing with a small amount of propylene glycol dicaprylate/dicaprateas demonstrated in FIG. 7B where homogeneous suspension was maintainedfor rituximab powder in 25:75 propylene glycoldicaprylate/dicaprate:ethyl lactate mixture after 2-week ambientstorage. It was demonstrated that overall suspension performance can beimproved using a suspension vehicle mixture.

CONCLUSIONS

These examples demonstrated that the non-aqueous powder suspensionapproach was feasible for high monoclonal antibody concentration SCadministration. Dry powder preparation by spray-drying was scalableusing the high efficiency spray-drying process. The most importantparameter for overall suspension performance was determined to be thetype of suspension vehicle. Powder suspension in ethyl lactate displayedexcellent suspension injectability with a low glide force of <15 N via a27-gauge TW staked needle for antibody concentration as high as 333mg/mL (total powder concentration of 500 mg/mL). Without being bound byany one theory, low viscosity and injectability could be attributed tostrong particle-suspension vehicle interaction that preventsparticle-particle agglomeration into larger particle size in thesuspension. However, this mechanism did not support physical suspensionstability. Dry antibody particles had a higher tendency to settle out inthe ethyl lactate suspension than in propylene glycoldicaprylate/dicaprate. The approach of using suspension vehicle mixtureproved to be effective in improving overall suspension performance.

1. A suspension formulation comprising a spray dried monoclonal antibodyat a concentration of about 200 mg/mL or more suspended in a non-aqueoussuspension vehicle, wherein the viscosity of the suspension vehicle isless than about 20 centipoise at about 25° C., and wherein thenon-aqueous suspension vehicle comprises propylene glycoldicaprylate/dicaprate.
 2. The formulation of claim 1 wherein theviscosity of the suspension vehicle is less than about 10 centipoise. 3.The formulation of claim 2 wherein the viscosity of the suspensionvehicle is less than about 5 centipoise.
 4. The formulation of claim 1wherein the injection glide force of the formulation is about 20 newtonor less.
 5. The formulation of claim 4 wherein the injection glide forceof the formulation is about 15 newton or less.
 6. The formulation ofclaim 1 wherein the average particle size in the formulation is fromabout 2 microns to about 30 microns.
 7. The formulation of claim 6wherein the average particle size in the formulation is from about 2microns to about 10 microns.
 8. The formulation of claim 1 wherein theantibody concentration in the formulation is from about 200 mg/mL toabout 500 mg/mL.
 9. The formulation of claim 8 wherein the antibodyconcentration in the formulation is from about 200 mg/mL to about 350mg/mL.
 10. The formulation of claim 1 further comprising a saccharide.11. The formulation of claim 10 wherein the saccharide is trehalose orsucrose.
 12. The formulation of claim 10 wherein the molar ratio ofsaccharide:monoclonal antibody is from about 50 to about 400:1.
 13. Theformulation of claim 12 wherein the molar ratio of saccharide:monoclonalantibody is from about 100 to about 250:1.
 14. The formulation of claim1 further comprising a surfactant.
 15. The formulation of claim 14wherein the surfactant is polysorbate 20 or polysorbate
 80. 16. Theformulation of claim 1 wherein the formulation is suitable forsubcutaneous administration.
 17. The formulation of claim 1 wherein themonoclonal antibody is a full length monoclonal antibody.
 18. Theformulation of claim 17 wherein the monoclonal antibody is a human IgG1.19. The formulation of claim 1 wherein the monoclonal antibody is achimeric, humanized, or human antibody.
 20. The formulation of claim 1wherein the monoclonal antibody binds an antigen selected from the groupconsisting of: CD20, HER2, VEGF, IL6R, beta7, Abeta, HER3, EGFR, andIgE.
 21. The formulation of claim 20 wherein the antibody is rituximab,trastuzumab, or bevacizumab. 22-24. (canceled)
 25. A subcutaneousadministration device with the formulation of claim 1 therein.
 26. Thedevice of claim 25 which comprises a pre-filled syringe.
 27. A method ofmaking a suspension formulation comprising suspending a spray driedmonoclonal antibody in a non-aqueous suspension vehicle with a viscosityless than about 20 centipoise at about 25° C., wherein the monoclonalantibody concentration in the suspension formulation is about 200 mg/mLor more, and wherein the non-aqueous suspension vehicle comprisespropylene glycol dicaprylate/dicaprate.
 28. A method of making anarticle of manufacture comprising filling a subcutaneous administrationdevice with the formulation of claim
 1. 29. A suspension formulationcomprising a spray dried full length human IgG1 monoclonal antibody at aconcentration from about 200 mg/mL to about 400 mg/mL suspended in anon-aqueous suspension vehicle with a viscosity less than about 20centipoise at about 25° C., wherein the formulation has an averageparticle size from about 2 microns to about 10 microns, and injectionglide force less than about 15 newton, and wherein the non-aqueoussuspension vehicle comprises propylene glycol dicaprylate/dicaprate. 30.The formulation of claim 29 which further comprises saccharide whereinthe molar ratio of saccharide:monoclonal antibody is from about 100 toabout 250:1.
 31. The formulation of claim 29 wherein the antibody isrituximab, trastuzumab, or bevacizumab.
 32. The formulation of claim 1for use in treating a disease or disorder in a patient.
 33. Use of theformulation of claim 1 in the preparation of a medicament for treating apatient in need of treatment with the monoclonal antibody in theformulation.
 34. A method of treating a patient comprising administeringthe formulation of claim 1 to a patient in need of treatment with themonoclonal antibody in the formulation.
 35. The method of claim 34wherein the formulation is administered subcutaneously to the patient.36. The method of claim 34 wherein the formulation is administered by apre-filled syringe containing the formulation therein.